This invention relates to electrokinetic methods of separating mixtures of various substances, and more particularly to the method known as isoelectric focusing used in the separation of mixtures of amphoteric substances. Specifically, the present invention relates to the method of and apparatus for establishing stable pH gradients in isoelectric focusing.
Amphoteric substances are substances that can behave either as acids or as bases, depending upon the hydrogen ion concentration of the solution in which they are present. At low pH values, amphoteric substances tend to acquire net positive charges, while at high pH values, they tend to acquire net negative charges. At an intermediate pH value (which varies from substance to substance), amphoteric substances exhibit net zero charges, and are then said to be at their isoelectric points.
Proteins are an example of naturally occuring amphoteric molecules. In addition to amphoteric molecules, mixtures of larger particles, such as viruses, cells, and cell organelles also exhibit an amphoteric character. In particular, various components of blood, e.g., the several kinds of white blood cells, are amphoteric. It is therefore very useful to have methods of separating mixtures of amphoteric substances, as such methods may be useful as analytical tools in biochemical investigations, in clinical medicine as an aid in the diagnosis of disease, and as technique useful in the preparation of quantities of purified substances, such as the insulins, the interferons, and the like.
Isoelectric focusing is a recently introduced method of separating mixtures of amphoteric substances. (For example, see "Isoelectric Focusing", P. G. Righetti and J. W. Drysdale, Laboratory Techniques in Biochemistry and Molecular Biology, T. S. Work and E. Work, editors, North Holland Publishing Company, Amsterdam, 1976, vol. 5, p. 335, and "Isoelectric Focusing and Isotachophoresis", N. Catsimpoolas, editor, Annals of the New York Academy of Sciences, June 15, 1973, vol. 209.) Isoelectric focusing may be described briefly as follows: a solution containing a mixture of amphoteric substances to be separated is placed in a channel along which a pH gradient has been established, and along which an electric field is applied by appropriate anode and cathode means. The pH gradient is usually established such that the pH value increases in the direction from anode to cathode. Under the influence of the electric field, particles having a net positive charge will migrate in the direction of the cathode while particles having a net negative charge will migrate in the direction of the anode. Neutral particles will experience no migration due to the field. Thus, under the influence of both an electric field and a pH gradient, amphoteric particles will separate, with each species of particle migrating to and concentrating at that position along the channel that has the pH value corresponding to the isoelectric point of the particular particle.
A number of methods have been used to establish a pH gradient in isoelectric focusing apparatus. A common prior art method involves the use of a mixture of amphoteric molecules of low molecular weight known as carrier ampholytes. The carrier ampholytes employed in the mixture are selected to have their isoelectric points at different pH values and to have optimum buffering capacity at these values. When placed in a channel under the influence of an electric field, a solution of carrier ampholytes will come to a steady state in which the several kinds of molecules will be stacked along the channel according to their isoelectric points, thus establishing a pH gradient.
There are several disadvantages associated with this method of establishing a pH gradient. Firstly, a phenomenon known as cathodic drift occurs. Briefly, the steady state positions of the various carrier ampholyte molecules drift slowly in the direction of the cathode. Consequently, the positions to which the amphoteric molecules being assayed or separated also slowly drift in the direction of the cathode. To achieve good separation, a sufficient time interval must be allowed from the start of the process to permit the various amphoteric molecules in an initially homogeneous mixture to migrate to the pH zones corresponding to their various isoelectric points. However, it will be appreciated that cathodic drift of the carrier ampholyte (and the pH zones) tends to mix the already separated molecules. Thus, cathodic drift limits both the yield and the resolving power of the separation method.
In addition, to separate amphoteric molecules with only slightly different isoelectric points, it is advantageous to have a shallow pH gradient (i.e., a gradient over a small pH interval). However, the smallest practicable interval that may be achieved with carrier ampholytes is on the order of 0.5 pH units.
Another disadvantage associated with the use of carrier ampholytes arises because certain amphoteric molecules form complexes with the carrier ampholytes. These complexes are focused according to the isoelectric points of the complexes. Thus, a single molecular species may be focused at several different positions along the channel depending upon the number of different molecules of the carrier ampholyte with which it forms complexes. This complicates both analytical and preparatory uses of isoelectric focusing.
Additionally, if isoelectric focusing is to be used for the preparation of purified substances, the carrier ampholytes will have to be removed in a subsequent operation. As it may be of primary importance that these substances remain biologically active, it is desirable to keep the number of operations necessary for the purification process to a minimum. Therefore it is desirable to achieve the pH gradient without carrier ampholytes.
In addition, it is difficult to separate molecules of the carrier ampholytes from certain low molecular weight amphoteric species such as the short polypeptides, which represent an important class of compounds of biological interest. It is also difficult to distinguish between the carrier ampholytes and the short polypeptides with the usual analytical methods of staining and spectral analysis. Again it is therefore desirable to achieve the pH gradient without carrier ampholytes.
Further, carrier ampholytes are relatively expensive.
Previous methods have been proposed in which isoelectric focusing is performed without carrier ampholytes. One such method involves dividing the focusing channel into several compartments held at graded pH values. The compartments are separated by membranes that allow the passage of the mixture to be separated yet maintain the selected pH values. (See, for example, A. J. P. Martin and F. Hampson, Journal of Chromatography, volume 159, 1978, p. 101.) A disadvantage of this approach is that membranes so constructed as to allow the passage of large molecules and particles, as many of the amphoteric substances of interest are, usually produce electroendosmotic flows that tend to disrupt the pH gradient. An additional disadvantage of this technique is that the pH gradient so established is not a continuous function, but, rather, a series of discrete values. Thus, a large number of compartments are required to obtain the resolutions required for most analytical purposes, a consideration which severely limits such applications of this type of isoelectric focusing device.
Still other methods use the principle of steady state electrolysis of buffer solutions in conjunction with two equal and oppositely-directed fluid flows between anode and cathode compartments. Focusing occurs in a convection-free zone, typically a flowtight gel, powder paste, or the like, between the cathode and anode and separate from the flow streams. (See, for example, H. Rilbe, Journal of Chromatography, volume 159, 1978, p. 193, and U.S. Pat. No. 4,217,193.) It is particularly difficult with this method to maintain stable concentration gradients, especially in the presence of anti-convective media.